Filamentous fungi are a rich source of agriculturally and pharmaceutically important natural products. For example, the fungal polyketide statins, such as lovastatin and compactin, are among the most widely- prescribed drugs for the prevention and treatment of atherosclerosis by inhibiting cholesterol biosynthesis. While the biosynthetic origins of bacterial polyketides have been studied extensively and have led to the combinatorial biosynthesis of pharmaceutically important "unnatural" natural products, the mechanistic and structural features of the corresponding enzymes from fungi have not been explored extensively, and their biosynthetic potential has not been realized. The objective of this proposal is to bridge these important knowledge gaps and provide a multi-angled picture of the fungal polyketide biosynthesis. Using the lovastatin polyketide synthase (PKS) as a model, we will study numerous novel mechanistic features of fungal polyketide synthases. The long-term goal of the study is to understand and engineer the biosynthesis of fungal polyketides. The goal of this collaborative proposal between a bioengineering/biochemistry laboratory (Tang) and a structural biology laboratory (Tsai) is to obtain mechanistic and structural insights into the lovastatin PKS and establish in vivo and in vitro platforms to examine this family of biosynthetic enzymes. This proposal will evaluate the following hypotheses: 1) fungal PKS enzymes expressed from Escherichia coli will retain activity and can be reconstituted to synthesize polyketides both in vivo and in vitro; 2) the individual domains in a multidomain fungal megasynthase can be reconstituted as standalone proteins and will reveal the basis for the iterative properties of fungal PKS enzymes; and 3) Structure-guided mutations of individual fungal PKS domains will provide insight into the active site conformation, as well as novel protein-protein interactions. To address these hypotheses in a two-year period, we have defined the following three SPECIFIC AIMS: Aim 1. Reconstitute fungal polyketide biosynthesis in a heterologous host. Aim 2. Mechanistic analysis of fungal PKS enzymes. Aim 3. Structural analysis of fungal PKS domains. [unreadable] [unreadable] [unreadable]